The present invention is in the field of molecular biology and diagnostics. In particular, the invention relates to a diagnostic assay for human Matrix Gla-protein (xe2x80x9cMGPxe2x80x9d), and its use as a biomarker for vascular condition and vascular new formation.
Cardiovascular disease is one of the major life-threatening diseases in the Western society, but biomarkers to monitor the severity or the progression of the disease are presently not available. Also, the number of biochemically detectable risk factors (e.g. serum cholesterol, triglycerides, ApoE genotype) is surprisingly low.
Vitamin K is a cofactor in the posttranslational conversion of glutamate residues into y-carboxyglutamate (Gla). At this time 10 mammalian Gla-containing proteins have been described in detail, and the number of Gla-residues per molecule varies from 3 (osteocalcin) to 13 (protein Z). In all cases in which their function was known, the activity of the various Gla-proteins was strictly dependent on the presence of the Gla-residues (Shearer, M. J., Brit. J. Haematol. (1990) 75:156-162; Vermeer, C., Biochem. J. (1990) 266:625-636). Gla-proteins are synthesized in various tissues, for instance the liver, bone and vessel wall. Blood coagulation factors 11 (prothrombin), VII, IX and X are examples of Gla-proteins synthesized in the liver, examples of so-called extrahepatic Gla-proteins are osteocalcin and Matrix Gla-Protein (Hauschka, P. V. et al., Phys. Rev. (1989) 69:990-1047).
Matrix Gla-Protein is a vitamin K-dependent protein synthesized in bone and in a number of soft tissues including heart and vessel wall. In experimental animals its soft tissue expression is high immediately after birth, but decreases in the months thereafter. Only in cartillage and arteries its expression seems to continue lifelong. Although the precise function of MGP on a molecular level has remained unknown so far, experiments with MGP-deficient transgenic animals (xe2x80x9cknock-outxe2x80x9d mice) have shown that MGP has a prominent role in the prevention of vascular mineralization: MGP-deficient animals were born to term but developed severe aortic calcification (as analyzed by X-ray) in the first weeks of life; eventually all animals died within 6-8 weeks after birth due to rupture of the aorta or one of the other main arteries (Luo, G. et al., Nature (1997) 386:78-81).
MGP was discovered in bone (Price. P. A. et at., Biochem. Biophys. Res. Commun. (1983) 117:765-771), but in situ hybridization experiments showed that it is also expressed in other tissues including the vessel wall (Fraser, D. J. et al., J. BioL Chem. (1988) 263:11033-11036). With polyclonal antibodies raised against a synthetic peptide homologous to the C-terminus of bovine MGP the protein was also found in cartilage via immunohistochemical staining (Loeser, R. F. et al., Biochem. J. (1992) 282:1-6). A radioimmunoassay was developed for the detection of serum MGP in the rat, but in these experiments circulating MGP was correlated with maturation of rat bone, and not with vascular biology (Otawara, Y. et al, J. Biol. Chem. (1986) 261:10828-10832).
Research concerning the role of MGP in the vessel wall has not started before the discovery by Luo et at (supra) that MGP is a strong inhibitor of vascular calcification in mice. Since then evidence has accumulated suggesting that bone calcification and atherosclerotic vessel wall calcification proceed via very similar mechanisms, in which the same proteins (including MGP) are used (Proudfoot, D. et al, Arterioscier. Thromb. Vasc. Biol. (1998) 18:379-388; Proudfoot, D. et al., J. Pathol. (1998) 185:1-3). Most studies on the regulation of MGP expression have been performed in smooth muscle cell cultures, with mRNA detection as a measure for MGP synthesis. Recent studies in humans have shown that, although MGP mRNA is constitutively expressed by normal vascular smooth muscle cells, it is substantially upregulated in cells adjacent to both medial and intimal calcification (Shanahan, C. M. et al., Cnt. Rev. in Eukar. Gene Expr. (1998) 8:357-375).
Dhore, C et al., Faseb J. (1999) 13, p. A203, disclose that MGP is produced in atherosclerotic plaques. The reference neither teaches nor suggests that MGP might enter the bloodstream from the plaque.
Sadowski, J. A. and O""Brien, M., Faseb J. (1991) 5, p. A944, disclose the production of polyclonal antibodies to a vitamin K-dependent epitope of MGP. The method is very general and can be used to any protein. No specific use of such antibodies for the assessment of MGP in human serum was suggested.
Price, P. A. et al., Arteroscier. Thmmb. Vasc. Biol. (1998) 18:1400-1407 disclose artificial calcification in a rat model system. This system is not a model for atherosclerosis, because it is not associated with vascular inflammation and plaque formation. The conclusion obtained in this model that vascular calcificsfion is associated with decreased serum MGP levels, is opposite to the findings according to the present invention in atherosclerotic patients, i.e. increased serum MGP in atherosclerosis.
The prior art does neither teach or suggest the use of MGP as a marker or angiogenesis or cardiovascular disease, or the like, nor does it disclose an assay for circulating human MGP.
As stated above, biomarkers to monitor the severity or the progression of cardiovascular disease are not available up till now, and the number of is biochemically detectable risk factors is very low. Therefore, there is clearly a need for biomarkers for vascular characteristics, for assessment of the severity or progression of atherosclerosis and related diseases, as well as for monitoring the effect of treatment during vascular disease.
In accordance with an aspect of the present invention, the use of a diagnostic assay is provided for the detection and determination of MGP in a human serum sample, the assay comprising the use of one or more antibodies, in particular monoclonal antibodies, specifically recognising epitopes on and/or conformations of human Matrix Gla-Protein.
The invention further provides a method for the production of such antibodies.
In another aspect of the invention, the assay comprises the use of said antibodies for the detection of any Matrix Gla-Protein in human serum as the total immunoreactive antigen, or for the detection of Matrix Gla-Protein in human serum as the fraction of carboxylated Matrix Gla-Protein (=5 Gla-residues/mol), or for the detection of Matrix Gla-Protein in human serum as the fraction of under-carboxylated MGP (xe2x89xa64 Gla-residues/mol).
In still a further aspect of the invention, a method is provided for using MGP-related antigens as biomarkers for certain diseases, for example, atherosclerosis and other vascular diseases, and angiogenesis/neogenesis in tumor development.
In a preferred embodiment of the invention, monoclonal antibodies of class IgG are provided for use in said diagnostic immunoassay which are obtainable by hybridomas formed by fusion of cells from a mouse myeloma and spleen cells from a mouse previously immunized with a peptide homologous to certain human MGP residues, in particular one of human MGP residues 3-15, human MGP residues 35-49, and human MGP residues 54-84, which antibodies are also referred to herein as mAb3-15, mAb35-49, and mAb54-84, respectively. Of these, mAb3-15 and mAb35-49 are preferred antibodies.
These and other aspects of the present invention will be more fully outlined in the detailed description which follows.